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一组用于酿酒酵母功能分析的表位标签整合载体。

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae.

作者信息

Sung Hyeran, Chul Han Kyung, Chul Kim Jun, Wan Oh Ki, Su Yoo Hwan, Tae Hong Jin, Bok Chung Youn, Lee Chong-Kil, Lee Kyung S, Song Sukgil

机构信息

College of Pharmacy, Chungbuk National University, 48 Gaeshindong, Cheongju, Chungbuk, Republic of Korea.

出版信息

FEMS Yeast Res. 2005 Jul;5(10):943-50. doi: 10.1016/j.femsyr.2005.03.008.

Abstract

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.

摘要

酿酒酵母基因的功能分析一直是基因组序列确定后的主要目标。尽管已经开发了几种用于分子遗传学分析的工具,但只有有限数量的可靠遗传工具可用于支持蛋白质水平的功能测定。表位标签是一种用于蛋白质检测、纯化和功能研究的强大工具。但是,利用整合载体开发的系统标签系统尚不存在。在此,我们构建了一组整合载体,可使感兴趣的蛋白质与四种不同的表位标签(HA、Myc、Flag和GFP)进行翻译融合。为了确认C末端标签蛋白的功能和表达,我们使用了Cdc11,它是 septin 丝的一个组成部分,环绕母芽颈,由五种主要蛋白质组成:Cdc3、Cdc10、Cdc11、Cdc12和Sep7。在其内源启动子下表达的Cdc11标签版本被发现具有生理功能,这通过其在颈部的定位以及与cdc11-6温度敏感突变相关的生长缺陷的抑制得到证明。用针对Cdc11或表位的抗体可以有效地检测到表达的蛋白质。当用抗Myc抗体进行免疫沉淀时,每个用Myc标签的septin蛋白都能与其他septin成分有效共纯化,表明形成了稳定的septin复合物。由于标签模块位于含有四种不同标记(HIS3、TRP1、LEU2或URA3)的整合载体上相同的18个限制酶切位点阵列下,该标签系统提供了高效的多重标签和目的基因的稳定表达。

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